The extensive application of conventional antibiotics has served as a selection pressure to drive the evolution of multiple drug resistant strains, Trevor et al., Curr. Opin. Infect. Dis. 18: 300-305, 2005. The rapid emergence of antibiotic-resistant bacteria, including those that are resistant to “last resort” antibiotics such as vancomysin, is one of the greatest challenges facing modern medicine and has provoked the exploration for new antimicrobial agents and strategies. A new paradigm in the treatment of infections is through activation of the innate immune system rather than, or in addition to, direct attack on the microbe as employed by conventional antibiotics, Finlay et al., Nat. Rev. Microbiol. 2: 497-504, 2004.
Host defense peptides (HDPs) have an evolutionary history of combating infections and represent a conserved mechanism of innate immune defense that is present in virtually all forms of life. In general HDPs range in size from twelve to fifty amino acids, carry a net positive charge and consist of approximately 50% hydrophobic residues. Hancock et al., Trends Microbiol. 8: 402-410, 2000. These peptides are an essential component of immune defense as their absence through either genetic knock-out or disease renders the host more susceptible to infections. Nizet et al., Nature 414: 454-457, 2001; Wilson et al., Science 286: 113-117, 1999; Ganz et al., J. Clin. Invest. 82: 552-556, 1988; Ong et al., N. Engl. J. Med. 347: 1151-1160, 2002; Putsep et al., Lancet 360: 1144-1149, 2002. Conversely HDP administration in animal models can prevent and treat bacterial infections, while offering protection from sepsis. Song et al., Antimicrob Agents Chemother. 49: 3868-74, 2005; Kwakman et al.; Antimirob Agents Chemother. 50: 3977-83, 2006, Giacometti et al., Crit. Care Med. 32: 2555-2556, 2004; Fukumoto et al., Pediatr. Surg. Int. 21: 20-24, 2005; Cirioni et al., Antimicrob. Agents Chemother. 50: 1672-1679, 2006.
The initial appreciation of the therapeutic potential of HDPs was from the perspective of their direct antimicrobial activity. While the specific details of direct antimicrobial action have yet to be conclusively determined, it is hypothesized that the majority, although not all, of HDPs kill bacteria through interactions with the bacterial membrane that ultimately lead to cell lysis. Subbalakshmi et al., FEMS Microbiol Lett. 160: 91-96, 1998; Brotz, H. et al., 42: 154-160, 1998, McPhee et al., J. Pept. Sci. 11: 677-687, 2005. That such a diverse range of HDPs have antimicrobial activity against a broad spectrum of bacteria, including Gram-negative and Gram-positive strains, suggests that the antimicrobial mechanism is dependent upon general characteristics of these molecules and their targets. Specifically, the positive charge of HDPs is hypothesized to be an essential feature in targeting the action of these molecules against bacterial membranes whose outer leaflet is rich in negatively-charged phospholipids, as opposed to the outer leaflet of plant and animal membranes which favor neutral lipids, McPhee et al., J. Pept. Sci. 11: 677-687, 2005.
There has been an emerging appreciation however that the biological action, and perhaps therapeutic potential, of these molecules extends beyond the ability to disrupt bacterial membranes. Indeed the physiological significance of HDP antimicrobial activity has been called into question as levels of expression of many of these peptides are below their antimicrobial thresholds and this activity is often suppressed in serum and by physiological concentrations of monovalent and divalent ions. Bowdish et al., Curr. Protein Pept. Sci. 6: 1-17, 2005; Bowdish et al., Antimicrob. Agents Chemother. 49: 1727-1732, 2005. Furthermore that peptides that do not possess antimicrobial activity still offer protection from bacterial challenges in animal models indicates that these molecules counter infections by influencing host cell process rather than direct antimicrobial activity. Scott et al., Nature Biotech. 25: 465-472, 2007; published online Mar. 25, 2007.
Emerging evidence suggests that HDPs assist in clearing infections by activating host cell processes associated with innate immunity, adaptive immunity and inflammation. HDPs have been shown to have involvement in a broad range of biological effects associated with immune functions including the up-regulation of chemokines/chemokines and their receptors, recruitment of leukocytes to sites of infection, stimulation of histamine release from mast cells, angiogenesis, dendritic cell maturation and wound healing. Bowdish et al., Curr. Protein Pept. Sci. 6: 1-17, 2005; Bowdish et al., Antimicrob. Agents Chemother. 49: 1727-1732, 2005; Hancock et al., Nat. Biotech. 24: 1551-1557, 2006; Scott et al., J. Immunol. 169: 3883-3891, 2002; Heilborn et al., J. Invest. Dermatol. 120: 379-389, 2003; Marr et al., Curr. Opin. Pharm. 6: 468-472, 2006. HDPs have also been shown to function as anti-endotoxic agents by modulating the deleterious consequences of inflammation and limiting the development of sepsis, Giacometti et al., Crit. Care Med. 32: 2555-2556, 2004; Fukumoto et al., Pediatr. Surg. Int. 21: 20-24, 2005; Cirioni et al., Antimicrob. Agents Chemother. 50: 1672-1679, 2006; Zasloff, Nature 415: 389-395, 2002.
While HDPs have found application in the treatment of topical infections (gramicidin S and polymyxin B), and as food preservatives (nisin), they have yet to achieve success in the larger market of treatment of systemic infections. One of the key limitations to the development of therapeutic HDPs for systemic treatment of bacterial infections has been their prohibitive cost. For example, synthesis of one gram of a typical HDP, which is the average daily dose required for systemic HDP administration, costs in the range of $100-$600. Hancock et al., Nat. Biotech. 24: 1551-1557, 2006. By comparison production of an equal quantity of an aminoglycoside antibiotic costs approximately 80 cents. Marr et al., Curr. Opin. Pharm. 6: 468-472, 2006. The high cost of peptide synthesis is compounded by the sensitivity of these molecules to proteolytic degradation as the biological half-life of HDPs measures in minutes. Finlay et al., Nat. Rev. Microbiol. 2: 497-504, 2004. Peptide modifications that increase biological stability to reduce dose quantities and frequencies will result in corresponding decreases in the associated costs. One such strategy of peptidomimetic optimization is through retro-inversion.
Retro-inversed (RI) peptides are isomers of natural peptides in which the sequence is reversed and the chirality of each amino acid is inverted, Chorev et al., TIBS 13: 438-445, 1995; Fischer, Curr. Prot. and Pept. Sci. 4: 339-356, 2003. Peptides modified in this manner are predicted to maintain the same three-dimensional topology of side-chains as their natural counterparts and therefore have the potential to maintain the same biological activities. A key structural and functional distinction of the retroinversed molecules is that peptide bonds linking D-amino acids are poor substrates for proteolytic enzymes which greatly improves the biological stability of RI-peptides, Fauchere et al., Adv. Drug. Res. 23: 127-159, 1992.
The application of retro-inversion to biological peptides has been explored with largely mixed results, Chorev et al., TIBS 13: 438-445, 1995; Fischer, Curr. Prot. and Pept. Sci. 4: 339-356, 2003. The differential ability of RI-peptides to maintain the functional characteristics of their natural counterparts is likely a consequence of the structural complexity of individual peptide as well as the mechanism through which it exerts its biological action. For peptides whose biological functions are dependent upon interactions with chiral molecules, such as DNA or protein, the extent to which the main-chain peptide groups contribute to complex formation will likely predict the extent to which the modification will be tolerated.
Retroinversion of HDPs has been explored in a limited number of cases with the general conclusion that the modification results in retained or moderately improved antimicrobial activity, Merrifield et al., Proc. Natl. Acad. Sci. USA 88: 4240-4244, 1995. Importantly however these investigations were limited to consideration of antimicrobial, rather than immunomodulatory, activity. As antimicrobial activity is mediated through non-stereospecific interactions with bacterial membranes it would be anticipated to be much more tolerant to retroinversion. The more stringent criteria by which retroinversion will be evaluated in HDPs is through the ability to influence higher-order innate immune function through interaction with chiral host receptors.
The extensive application of conventional antibiotics has served as a selection pressure to drive the evolution of multiple drug resistant strains of bacteria and other infectious agents that can cause infectious disease. A need exists in the art for improved and effective therapeutic compounds for treatment of infectious diseases, e.g., bacterial and parasitic infectious disease, and inflammatory diseases.